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Maren Depke Discussion and Conclusions explained by the missing pathogenic antigen processing in dendritic cells and the consequently missing activation of specific cytotoxic CD8 + T cells (Tu et al. 2009). The induction of immunoproteasome β i -subunits but also of Tap1 was reported to be mediated by STAT1 during signal transduction and IRF-1, which acts as transcriptional activator (White et al. 1996, Chatterjee-Kishore et al. 1998, Foss/Prydz 1999, Brucet et al. 2004, Namiki et al. 2005). In agreement with literature information, IFN-γ treatment led to a clear induction of STAT1 and IRF-1 in BMM of both strains in the study of murine BMM described in this thesis (data not shown). The gene expression profiling revealed the influence of IFN-γ on several cytokines. BMM induced several chemokines (Ccl5, Ccl12, Ccl22, Cxcl9, Cxcl10, Cxcl11, Cxcl16), chemokine receptors (Ccr5, Ccrl2), interleukins (Il15, Il27), interleukin receptors (Il2rg, Il4ra, Il10ra, Il12rb1, Il13ra1, Il15ra, Il21r) and also TNF and TNF family ligands (Tnf, Tnfsf10, Tnfsf18) and receptor (Tnfrsf14). These were interpreted as preparation for a potentially following second stimulus and consequent immune reaction, which includes pro- and anti-inflammatory directions. Cxcl9, Cxcl10, and Cxcl11 are known to be induced by IFN-γ (Luster et al. 1985, Farber 1990, Taub et al. 1993, Liao et al. 1995, Cole KE et al. 1998). These chemokines bind the receptor Cxcr3, which is preferentially found on IL-2 activated Th1 cells (Loetscher M et al. 1996). Of these three chemokines, Cxcl11 has highest affinity to Cxcr3 and additionally is the most potent agonist (Cole KE et al. 1998). Because the cytokine exerts its influence only on activated T cells, the authors additionally infer that the cytokine mainly acts during the immune response and aims to direct effector T cells to the manifestation site, when T cell activation, IL-2 production, and proliferation of specific T cells had occurred in lymphoid organs (Cole KE et al. 1998). The three chemokines Cxcl9, Cxcl10, and Cxcl11 exert their function not only in the chemotaxis of Th1 cells via Cxcr3. It was reported that they also can bind to Ccr3, the receptor for several Ccl chemokines (Ccl5, Ccl7, Ccl8, Ccl11, Ccl13, Ccl24), which is found on Th2 cells. Cxcl9, Cxcl10, and Cxcl11 act antagonistic when binding to Ccr3. Thus, these chemokines do not only attract Th1 cells but also inhibit chemotaxis of Th2 cells and further polarize the immune response (Loetscher P et al. 2001). More recently, a similar antagonistic effect of Cxcl11 on the receptor Ccr5 was reported (Petkovic et al. 2004). Most interestingly, beside agonist and antagonist functions, Cxcl9, Cxcl10, and Cxcl11 have been reported to exhibit direct antimicrobial functions against Escherichia coli and Listeria monocytogenes. The observation was rated as biologically relevant since antimicrobially effective concentrations of chemokines were determined in vitro and in vivo (Cole AM et al. 2001). The antimicrobial effect of Cxcl9, Cxcl10, and Cxcl11 also applied to S. aureus (Yang D et al. 2003). Another study determined constitutive expression of Cxcl9 in the human male reproductive system and secretion into seminal plasma where antibacterial activity against the urogenital pathogen Neisseria gonorrhoeae was demonstrated leading to the conclusion that Cxcl9 is relevant for the host’s local immune defense (Linge et al. 2008). The multifunctional characteristic of chemokine and antimicrobial function is not a unique feature of Cxcl9, Cxcl10, and Cxcl11, but was observed for several other chemokines, too. Antimicrobial activity could be assigned to about two third of all studied chemokines (Yang D et al. 2003). The cytokine TNF was induced in BMM after IFN-γ treatment. TNF is the central inflammation mediator. Since long it is known that TNF is the mediator of lethal endotoxin effects (Beutler et al. 1985), which finding was worth a reprint as part of the “Pillars of Immunology” series in The Journal of Immunology (Vilcek 2008). In the meantime, knowledge was expanded and 184
Maren Depke Discussion and Conclusions superfamilies of TNF ligands and receptors were identified (Hehlgans/Pfeffer 2005). TNF is associated with several functions linked to pro-inflammatory effects, which involve a multiprotein signaling complex at the cell membrane and MAP-kinase signaling and which are finally mediated via NFκB and c-Jun (Chen G/Goeddel 2002, Wajant et al. 2003). Rarely, TNF can induce apoptosis via caspase activation, depending on the cellular balance of antiapoptosis/proliferation signals mediated by NFκB (Gaur/Aggarwal 2003, Wajant et al. 2003). Furthermore, TNF is involved in the regulation of fever with both pyrogenic and antipyretic effects depending on physiological or experimental conditions (Leon 2002). In macrophages, TNF has an important function in controlling intracellular mycobacterial growth (Bekker et al. 2001). TNF deficient mice are susceptible to Listeria monocytogenes infections, exhibit reduced contact hypersensitivity and impairment of splenic follicular architecture and of maturation of the humoral immune response (Pasparakis et al. 1996). Knockout of TNF and lymphotoxin-α (TNF-β) leads to an increased susceptibility to S. aureus infections, but also to a reduced frequency of arthritis indicating a damaging influence of the cytokine during the inflammatory reaction (Hultgren et al. 1998). Reflecting the central position of TNF in the regulation of the immune response to infection, several strategies of pathogens to influence the TNF activity were reported. The interference can lead to diverse and contrary effects depending on the pathogen, like inhibition of apoptosis, enhancement of apoptosis, inhibition of TNF production or, in case of S. aureus, the induction of a TNF-like response by interaction of protein A with the TNF receptor TNFR1 (Rahman/McFadden 2006). Beside other functions, TNF was described to further enhance the expression of the immunoproteasome (Loukissa et al. 2000). In non-professional antigen-presenting cells, the induction of immunoproteasome, TAP peptide transporters, and MHC class I complexes by TNF independent of IFN-γ activity was observed and in addition increased stability of the MHC class I complexes at the cell surfaces (Hallermalm et al. 2001). Thus, induction of TNF in BMM after IFN-γ treatment might intensify the effect on antigen processing and presentation after a second stimulus. Also in the serum-free system, the inducibility of TNF in IFN-γ and LPS treated BMM was described (Eske et al. 2009). IL-6, IL-10, and IL-12, which were also inducible by IFN-γ and LPS (Eske et al. 2009), were not expressed in IFN-γ treated BMM (this study), and the IFN-γ and LPS inducible chemokine Ccl2 (MCP-1; Eske et al. 2009) was expressed but not differentially regulated in IFN-γ treated BMM (this study). This strongly hints for the explanation that the induction only occurs when a second stimulus like LPS is given. After IFN-γ treatment, the induction of anti-inflammatory IL-10 receptor (Moore et al. 2001) as well as the induction of Il18bp coding for an IL-18 binding protein, which is a natural endogenous inhibitor of the proinflammatory IL-18 (McInnes et al. 2000, Gracie et al. 2003, Novick D et al. 1999), was observed (this study). Not only Stat1 and Irf1 of the positive IFN-γ feedback were induced, but also Stat3 and Socs1 which are implicated in negative feedback (Schroder et al. 2004, Hu et al. 2008). This might indicate the possible reactivity to antiinflammatory stimuli and a confinement of inflammatory response. After the priming signal of IFN-γ, the BMM are not fully activated and are not expected to secrete cytokines, but rather after a second stimulus (Hu et al. 2008). Analysis of cytokine secretion of BMM beyond the examples analyzed until now will be of interest, either after stimulation with LPS, or after stimulation with molecules of Gram-positive bacteria or even infection e. g. with S. aureus, as it will be in focus of research for follow-up experiments to this study. 185
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G E N O M E W I D E C H A R A C T E
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Maren Depke C O N T E N T S GENOMEW
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Maren Depke Z U S A M M E N F A S S
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Maren Depke Zusammenfassung der Dis
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Maren Depke S U M M A R Y O F D I S
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Maren Depke Summary of Dissertation
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Maren Depke I N T R O D U C T I O N
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Maren Depke Introduction Besides op
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Maren Depke Introduction Extracellu
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Maren Depke Introduction 2005). SaP
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Maren Depke Introduction contains a
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Maren Depke Introduction via fibron
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Maren Depke Introduction cathelicid
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Maren Depke Introduction STUDIES OF
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Maren Depke Introduction are effect
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Maren Depke Introduction cell stimu
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Maren Depke Introduction channel CF
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Maren Depke M A T E R I A L A N D M
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Maren Depke Material and Methods Li
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Maren Depke Material and Methods Ki
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Maren Depke Material and Methods GE
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Maren Depke Material and Methods Pa
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corticosterone [pg/ml plasma] corti
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Maren Depke Results Liver Gene Expr
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lood glucose levels [nmol/l] resist
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LBP [ng/ml] CPR [ng/ml] Maren Depke
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Maren Depke Results Liver Gene Expr
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liver weight [g] Maren Depke Result
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infection rate cfu / 10 mg tissue c
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Maren Depke Results Kidney Gene Exp
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Table R.2.1: Genes displaying stati
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Maren Depke BR1 sign DsigB vs wt BR
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Maren Depke Results GENE EXPRESSION
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Maren Depke Results Gene Expression
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log2(ratio C57BL/6 / BALB/c) at IFN
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Maren Depke Results Host Cell Gene
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Maren Depke Results Pathogen Gene E
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Page 218 and 219: Maren Depke References Jin T, Bokar
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Page 226 and 227: Maren Depke References Puccetti P.
Page 228 and 229: Maren Depke References Siewert E, B
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Maren Depke A F F I D A V I T / E R
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Maren Depke Acknowledgments Kidney
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